There is a continuous and expanding need for rapid, highly specific methods of detecting and quantifying chemical, biochemical and biological substances as analytes in research and diagnostic mixtures. Of particular value are methods for measuring small quantities of nucleic acids, peptides, pharmaceuticals, metabolites, microorganisms and other materials of diagnostic value. Examples of such materials include small molecular bioactive materials (e.g., narcotics and poisons, drugs administered for therapeutic purposes, hormones), pathogenic microorganisms and viruses, antibodies, and enzymes and nucleic acids, particularly those implicated in disease states.
The presence of a particular analyte can often be determined by binding methods that exploit the high degree of specificity which characterize many biochemical and biological systems. Frequently used methods are based on, for example, antigen-antibody systems, nucleic acid hybridization techniques, and protein-ligand systems. In these methods, the existence of a complex of diagnostic value is typically indicated by the presence or absence of an observable “label” which has been attached to one or more of the interacting materials. The specific labeling method chosen often dictates the usefulness and versatility of a particular system for detecting an analyte of interest. Preferred labels are inexpensive, safe, and capable of being attached efficiently to a wide variety of chemical, biochemical, and biological materials without significantly altering the important binding characteristics of those materials. The label should give a highly characteristic signal, and should be rarely, and preferably never, found in nature. The label should be stable and detectable in aqueous systems over periods of time ranging up to months. Detection of the label is preferably rapid, sensitive, and reproducible without the need for expensive, specialized facilities or the need for special precautions to protect personnel. Quantification of the label is preferably relatively independent of variables such as temperature and the composition of the mixture to be assayed.
A wide variety of labels have been developed, each with particular advantages and disadvantages. For example, radioactive labels are quite versatile, and can be detected at very low concentrations. Such labels are, however, expensive, hazardous, and their use requires sophisticated equipment and trained personnel. Thus, there is wide interest in non-radioactive labels, particularly in labels that are observable by spectrophotometric, spin resonance and luminescence techniques, and reactive materials, such as enzymes that produce such molecules.
Labels that are detectable using fluorescence spectroscopy are of particular interest, because of the large number of such labels that are known in the art. Moreover, the literature is replete with syntheses of fluorescent labels that are derivatized to allow their facile attachment to other molecules, and many such fluorescent labels are commercially available.
In addition to being directly detected, many fluorescent labels operate to quench the fluorescence of an adjacent second fluorescent label. Because of its dependence on the distance and the magnitude of the interaction between the quencher and the fluorophore, the quenching of a fluorescent species provides a sensitive probe of molecular conformation and binding, or other, interactions. An excellent example of the use of fluorescent reporter quencher pairs is found in the detection and analysis of nucleic acids.
Conventional organic fluorophores generally have short fluorescence lifetimes, on the order of nanoseconds (ns), which is generally too short for optimal discrimination from background fluorescence. An alternative detection scheme, which is theoretically more sensitive than conventional fluorescence, is time-resolved luminescence. According to this method, a chelated lanthanide metal with a long radiative lifetime is attached to a molecule of interest. Pulsed excitation combined with a gated detection system allows for effective discrimination against short-lived background emission. For example, using this approach, the detection and quantification of DNA hybrids via an europium-labeled antibody has been demonstrated (Syvanen et al., Nucleic Acids Research 14: 1017-1028 (1986)). In addition, biotinylated DNA was measured in microtiter wells using Eu-labeled streptavidin (Dahlen, Anal. Biochem. (1982), 164: 78-83). A disadvantage, however, of these types of assays is that the label must be washed from the probe and its luminescence developed in an enhancement solution.
In view of the predictable practical advantages it has been generally desired that the lanthanide chelates employed should exhibit a delayed luminescence with decay times of more than 10 μs. The luminescence of many of the known luminescent chelates tends to be inhibited by water. As water is generally present in an assay, particularly an immunoassay system, lanthanide complexes that undergo inhibition of luminescence in the presence of water are viewed as somewhat unfavorable or impractical for many applications. Moreover, the short luminescence decay times is considered a disadvantage of these compounds. This inhibition is due to the affinity of the lanthanide ions for coordinating water molecules. When the lanthanide ion has coordinated water molecules, the absorbed light energy (excitation energy) is quenched rather than being emitted as luminescence.
Thus, lanthanide chelates, particularly coordinatively saturated chelates that exhibit excellent luminescence properties are highly desirable. Alternatively, coordinatively unsaturated lanthanide chelates exhibiting acceptable luminescence in the presence of water are also advantageous. Such chelates that are derivatized to allow their conjugation to one or more components of an assay find use in a range of different assay formats. The present invention provides these and other such compounds and assays using these compounds. Hydroxyisophthalamide (IAM) complexes of lanthanide ions such as Tb3+ are potentially useful in a variety of biological applications. Of particular importance for biological applications is that these complexes exhibit kinetic stability in aqueous solutions at concentrations at or below nM levels.

Hydroxyisophthalamide ligands useful in applications requiring luminescence have been described (Petoud et al., J. Am. Chem. Soc. 2003, 125, 13324-13325; U.S. Pat. No. 7,018,850 to Raymond et al.). The H(2,2) backbone has been employed to synthesize isophthalamide-based ligands such as 1 and 2 in FIG. 1. Those octadentate ligands display relatively high thermodynamic stability when chelated to trivalent lanthanide ions. The functionalized TIAM ligand 2 has been conjugated to biomolecules and used as a donor in TR-LRET studies (Johansson et al., J. Am. Chem. Soc. 2004, 126(50):16451-16455).
However, a need for luminescent complexes, which are stable under biologically relevant conditions and at low concentrations, and which simultaneously exhibit low non-specific interactions with proteins, remains. The current invention addresses these and other needs.